1. Field of the Invention
This invention relates to symplectin, which is a photoprotein derived from okinawan squid (Symplectoteuthis oualaniensis: Tobi-Ika). Moreover, this invention relates to an amino acid sequence of symplectin and a base sequence of gene encoding the protein. Furthermore, the present invention relates to a method for detection of a monovalent cation by monitoring luminescence, generated using said photoprotein in the presence of a chromophore.
2. Prior Art
Bioluminescence has been applied for various purpose, such as monitoring of concentration of a metal ion in a living cell, because some complementary factor is requisite for generation of luminescence. A chromophore is oxidized and then a high energy state intermediate (luminescent intermediate) is formed. It collapses to form a basal state and emission of luminescence occurs accompanied with such alteration of energy state. In a photogenic organism, emission of luminescent occurs efficiently using an enzyme.
The most classical bioluminescence is observed in firefly, which is well-known as luciferin-luciferase reaction. In luciferin-luciferase reaction, luciferin is converted to oxyluciferin via enzymatic reaction of luciferase in the presence of ATP and magnesium ion. It is the essential phenomenon involved in the photo-reaction. As the reaction mediated by firefly luciferase enables detection of luminescence with extremely high sensitivity, it is an important tool for investigation in the field of biochemistry. The gene encoding firefly luciferase has been already cloned. Then, heat-stable type luciferase is produced by genetic engineering technique using E. coli and it is commercially available.
For another example, aequorin, a blue fluorescent protein of jellyfish (Aequorea victoria) capable of emitting blue color luminescence, has been also known. Aequorin has a relatively low molecular mass of 21 kDa. Aequorin uptakes a chromophore such as coelenterazine and oxygen, then it is converted to an exited state (high energy state) using calcium ion as a trigger, thereby emits blue color luminescence. The color of jellyfish luminescence is actually blue color and not green color. However, green fluorescent protein (GFP) is assumed to receive energy from aequorin, thereby emitting green color luminescence.
The above luminescent system of jellyfish has been applied in the field of cell physiology and biochemistry. Emission of luminescence caused by aequorin is triggered by calcium ion. Then aequorin has been utilized to detect alteration of calcium ion concentration, such as for analysis on alteration of cytoplasmic calcium ion concentration caused by agonistic stimulation of intact cells, for example, by various hormones, agonists such as neurotransmitters or growth factors. Therefore, aequorin has attracted attention as a calcium sensor or a gene reporter, in the field of clinical biochemistry and cell physiology.
The above-mentioned method, utilizing luciferin or aequorin, is an excellent method for its high sensitivity. However, the ions which serves as the trigger of luminescence are magnesium ion and calcium ion for luciferin and aequorin, respectively. These are both divalent cations. Thus, there has been no means which enables detection of a monovalent cation using a photoprotein. The method utilizing a photoprotein is advantageous, because its sensitivity is extremely high and it is free from danger like experiments using a radioisotope. Therefore, a photoprotein which can detect a monovalent cation has been desired.